The decision of whether and where to cross the midline, an evolutionarily conserved line of bilateral symmetry in the central nervous system, is the first task for many newly extending axons. Wnt5, a member of the conserved Wnt secreted glycoprotein family, is required for the formation of the anterior of the two midline-crossing commissures present in each Drosophila hemisegment. Initial path finding of pioneering neurons across the midline in both commissures is normal in Wnt5 mutant embryos; however, the subsequent separation of the early midline-crossing axons into two distinct commissures does not occur. The majority of the follower axons that normally cross the midline in the anterior commissure fail to do so, remaining tightly associated near their cell bodies, or projecting inappropriately across the midline in between the commissures. The lateral and intermediate longitudinal pathways also fail to form correctly, similarly reflecting earlier failures in pathway defasciculation. Panneural expression of Wnt5 in a Wnt5 mutant background rescues both the commissural and longitudinal defects. Wnt5 protein is predominantly present on posterior commissural axons and at a low level on the anterior commissure and longitudinal projections. Transcriptional repression of Wnt5 in AC neurons by the Wnt5 receptor, Derailed, contributes to this largely posterior commissural localization of Wnt5 protein (Fradkin, 2004).

The correct wiring of a nervous system requires that a large number of neurons stereotypically extend their axonal processes to make synaptic contacts with their muscle and neuronal targets. The leading portion of the axon, the growth cone, is faced with a bewildering number of routing decisions as it travels, frequently many hundreds of cell body diameters, to its target (Fradkin, 2004).

Path finding relies on the growth cone receiving and interpreting guidance cues presented to it at intermediate points in its journey. While the growth cone likely integrates multiple signals at many points, the initial extension of axons has received the most scrutiny; several of the important attractive and repulsive guidance cues and their neuronal receptors have been identified (Fradkin, 2004).

In Drosophila, the majority of the neurons of the embryonic ventral cord are born near the ventral midline, a morphological and functional line of symmetry whose vertebrate equivalent is the floorplate. Axon tracts in the mature embryo form a characteristic “ladder-like” structure reflecting the presence of two longitudinal tracts that extend in the anterior–posterior axis and two commissural tracts, the anterior (AC) and posterior commissures (PC) that bridge the longitudinal pathways in every segment (Fradkin, 2004).

The first choice for many newly extending axons is whether to cross the ventral midline. The axons of certain neurons, the ipsilaterals, do not cross the midline, but instead project with other longitudinal axons toward the anterior or posterior. Other axonal pathways, the contralaterals, cross the midline, extend along the longitudinal tracts and do not recross. The decision to cross or not and the prevention of repeated midline crossing are regulated by interactions between the extending axons and the midline glia cells (MG), specialized cells that emanate repulsive and attractive signals and underlie both the AC and PC at the ventral midline. The Netrin and Slit proteins are among the best characterized of the midline-derived cues. Netrins, signaling through axonal Frazzled receptors, primarily act as attractants, although repulsive Netrin-dependent signaling has also been reported. Slit protein signaling through the axonal Robo family of receptor proteins repels axons away from the midline, thus preventing them from recrossing. In addition to their midline roles, evidence has been provided that the expression domains of the three Robo proteins also delimit lateral domains within the longitudinal axon tracts (Fradkin, 2004).

Initial outgrowth and path finding of pioneering axons is, at least in part, dependent on both their interactions with the glial cell scaffold and their responsiveness to midline-derived cues. Subsequently, “follower” axons fasciculate with the pioneers to form multiaxon fascicles. Regulation of the relative balance between fasciculation and defasciculation through regulation of cell adhesion molecule activities at specific points allows individual axons to branch off to follow separate trajectories. Several cell adhesion proteins have been shown to act in the fasciculation of embryonic Drosophila axons. Embryos bearing mutations in the Drosophila NCAM ortholog, fasciculin II (fasII), display inappropriately defasciculated axons, whereas axons that overexpress fasII become hyperfasciculated. The Connectin (Conn) protein effects homophilic interactions between motoneurons. The beaten path (beat-Ia) gene encodes an Ig domain-containing protein secreted from axonal growth cones. In beat-Ia mutants, axons become hyperfasciculated in a manner suppressible by fasII and conn mutations, suggesting that Beat-Ia acts as a secreted anti-adhesive factor. There are 13 other Drosophila beat genes; interestingly, those four whose full ORF sequences have been determined encode transmembrane or GPI-linked proteins. Genetic interactions between beat-Ia and beat-Ic (encoding a transmembrane Beat protein) indicate their complementary functions, with beat-Ic and beat-Ia acting to increase and decrease adhesiveness, respectively. The Beat receptor(s) have not yet been identified (Fradkin, 2004).

Most of the mechanisms regulating guidance across the midline that have been uncovered thus far operate in both the AC and PC. Little is known about the mechanisms that underlie the choice of axons to go through either the AC or the PC. One gene implicated in this process is derailed (drl), a member of RYK subfamily of receptor tyrosine kinases. Recent studies have demonstrated that interactions between the Drl and Wnt5 proteins play an important role in preventing AC axons from inappropriately crossing in the PC (Yoshikawa, 2003). AC axons are less tightly fasciculated in the drl mutant, suggesting that Drl may act to regulate interneuronal adhesion (Fradkin, 2004).

Wnt5 is a member of the Wnt gene family, a large group of evolutionarily conserved genes encoding secreted glycoproteins that play roles at many developmental stages in a variety of tissues. Among other roles in the nervous system, Wnt proteins act in cell fate determination, synapse formation and maintenance and as mitogens (Fradkin, 2004).

Evidence has been provided that the Drosophila Wnt5 protein is found on the embryonic CNS axon tracts (Fradkin, 1995). The Wnt5 gene encodes a highly unusual Wnt protein that bears a long amino terminal extension to the Wnt-homologous domain that contains no known conserved domains. This report presents a detailed analysis of the Wnt5 mRNA and protein expression domains and demonstrates a crucial role for Wnt5 in formation of the major axon tracts during embryonic CNS development through examination of embryos lacking Wnt5. Wnt5 protein is required for the separation or defasciculation of early axonal projections that subsequently form the mature commissural and longitudinal connectives. Evidence is provided that the porcupine (porc) gene is a member of theWnt5 signaling pathway and that the Wnt5 gene is itself one of the downstream targets (Fradkin, 2004).

Wnt5 mRNA is most highly expressed by a large number of neurons predominantly in lateral and midline clusters underlying the PC, but is also found in a few cell bodies more closely associated with the AC. Wnt5 is not apparently expressed by the midline or lateral glia cells. While Wnt5 protein is detected on both AC and PC axons, as well as the longitudinal axon tracts, Wnt5 protein is expressed at higher levels on the PC as compared with the AC from the earliest stages when axons begin to extend and throughout embryonic CNS development (Fradkin, 2004).

Wnt5 null alleles were generated to determine the role of Wnt5 in CNS development. Early BP102⁺ commissural axons that pioneer first the PC, and subsequently the AC, are unaffected in the Wnt5 mutant. In the wild-type embryo, the BP102⁺ AC and PC pioneers and their early followers are closely associated near the midline, but subsequently separate at stage 13, to begin the formation of the two distinct commissures. This separation does not take place in the majority of Wnt5 mutant segments (Fradkin, 2004).

Visualization of the Sema2b⁺ axons, followers that cross in the wild-type AC at early stage 15, reveals that they largely fail to do so in the Wnt5 mutant and fasciculate inappropriately with ipsilateral sibling longitudinal projections or cross the midline in the region between the AC and PC. The PC axon trajectories were visualized by panneural staining and in the Eg⁺ PC-crossing lineage and no major alterations were seen in the Wnt5 mutant (Fradkin, 2004).

In addition to the commissural defects in Wnt5 mutant embryos, alterations to the longitudinal pathway projections were observed. Wnt5 mutant embryos display differential phenotypes with respect to the FasII⁺ longitudinal pathways: the medial or innermost pathway is largely unaffected; however, breaks are found in the intermediate pathway and the lateral pathways. Supporting these observations, the Ap⁺ projections, which in wild type follow the ipsilateral medial pathway, were unaffected by the absence of Wnt5. Visualization of the intermediate and lateral pathways with anti-Robo3 and anti-Robo2, respectively, further indicated that disruption became more severe in the pathways more lateral to the ventral midline (Fradkin, 2004).

Analyses of the longitudinal pioneer neurons indicate that, when specific axons have to selectively defasciculate to pioneer new pathways, particularly the intermediate and lateral pathways, they do not do so in the absence of Wnt5. Examination of the Robo2⁺ neurons indicates that after a limited period of extension, they stop and fail to form the continuous fascicle seen in the wild-type embryo. The Wnt5 longitudinal phenotypes are unlikely to simply reflect the failure of the axons that normally transit the AC to cross based on the following observations: (1) early defasiculation defects in the longitudinal pioneering projections at times where the commissures themselves are being pioneered, (2) discontinuities in ipsilaterally projecting pathways that do not cross the midline, and (3) apparently normal Fas2⁺ longitudinal pathways in the comm null mutant, where few if any axons cross in either commissure (Fradkin, 2004).

The VUM neurons, which normally aid in separating the commissures by sending their projections in between the PC and AC BP102⁺ pioneers, project abnormally in the Wnt5 mutant. Migration of the VUM cell bodies and the MG, which are also involved in establishing the physical separation between the AC and PC, do, however, occur normally in the Wnt5 mutant. Because the failure to establish the AC in the Wnt5 mutant occurs with a similar frequency (67%) to that observed for the abnormal VUM projections (70%), the VUM abnormalities likely contribute to the later failures of the follower axons to form the AC. The VUMs express Wnt5 mRNA at the time of commissure separationand may therefore represent the chief source of Wnt5 protein mediating separation of the AC and PC (Fradkin, 2004).

How does Wnt5 mediate the formation of the AC and the lateral longitudinal pathways? The data, including the observation that Wnt5 protein is most highly expressed on the PC, support the previously proposed role for PC-expressed Wnt5 as a repellent for Drl+ axons (Yoshikawa, 2003), but reveal the likely nature of Wnt5-mediated repulsion required to effect the formation of the AC and more lateral longitudinal pathways. It is suggested that the major role of Wnt5 is to mediate the selective defasciculation of the early commissural and longitudinal pathways necessary for them to separate into distinct commissures or pioneer new pathways, respectively. As defasciculation may be viewed as local repulsion or decreased attraction between axons, this interpretation of the early commissural defects in the Wnt5 mutant is therefore not at odds with Wnt5 acting as a PC-derived repellent. However, Wnt5 appears to act by facilitating the defasciculation of Drl⁺ axons from their siblings necessary for formation of the AC. The role of drl in the Wnt5 mutant longitudinal pathway defasciculation defects is presently unclear. Interestingly, studies of the drl mutant phenotype described the inappropriate defasiculation of axons lacking drl. Similar drl null phenotypes were observed as previously reported; however, the drl CNS phenotypes are consistently less severe than those seen in the Wnt5 null, suggesting that other genes may also interact with Wnt5 to effect selective fasciculation (Fradkin, 2004).

Tescue experiments demonstrated that the primary requirement for Wnt5 expression during embryonic CNS development is in neurons. Panneural Wnt5 expression rescued both the commissural and longitudinal defects. Strikingly, high levels of Wnt5 secreted throughout the CNS-driven by the Repo-Gal4 lateral glial cell driver in the Wnt5 mutant background fail to rescue, suggesting that neurons may process Wnt5 differently than the lateral glia. In support of this possibility, evidence has been presented that Wnt5 protein secreted from tissue culture cells and produced in embryos, respectively, is proteolytically processed (Fradkin, 2004).

Wnt5 overexpression at the AC midline (Sim-Gal4 driver), but not the PC midline (Btl-Gal4 driver) results in a failure to form the AC without noticeable effects on the PC. The noncrossing axons appear tightly associated in the longitudinals, suggesting that Wnt5 protein levels may be important in the regulation of axonal adhesion, with either too little or too much Wnt5 resulting in overly tight fasciculation. The Sim-Gal4-driven Wnt5 overexpression phenotype has also been interpreted to indicate that ectopic Wnt5 repulses all Drl⁺ AC axons (Yoshikawa, 2003). However, given the current absence of data showing that Wnt5 can directly collapse Drl⁺ growth cones, it is equally possible that the overly tight fasciculation of those axons precludes their midline crossing (Fradkin, 2004).

This study has shown that the differing levels of Wnt5 protein on the PC vs. AC are maintained, at least in part, from repression of Wnt5 transcription in AC neurons by Drl. Furthermore, apparently normal CNS architecture is observed when Wnt5 is pan-neurally expressed in a wild-type background (in Elav-GAL4 X UAS-Wnt5 embryos) resulting in high levels of Wnt5 protein on both commissures. The question arises as to why such regulation of Wnt5 in the AC exists? One possibility is that, although high levels of Wnt5 protein on both commissures result in rescue of the Wnt5 mutant phenotype and a lack of an observable phenotype in the wild-type background, respectively, the active species of Wnt5 protein may be asymmetrically generated with respect to the commissure of origin, reflecting PC- vs. AC-specific Wnt5 protein processing. Alternatively, the Drl-mediated repression of AC Wnt5 expression may reflect a requirement for low levels of Wnt5 in AC axons while precluding the higher levels that effect defasciculation, likely via the Drl receptor (Fradkin, 2004).

This study found that porcupine gene expression is required for the MG Wnt5 overexpression phenotype. Since porc aids in Wnt5 protein secretion (Tanaka, 2002), these data indicate porc-mediated Wnt5 secretion is required for Wnt5 signaling in this overexpression assay. porc function appears to be limiting for Wnt5 secretion in this assay as reduction of porc gene dosage by half in the porc/+ females is sufficient to suppress the Wnt5 midline overexpression phenotype. Is porc required for wild-type Wnt5-mediated signaling? The BP102⁺ axon tracts are disorganized in porc germline clone mutants lacking both maternal and zygotic porc (Tanaka, 2002); however, interpretation of this phenotype is complicated by the requirement for porc in wg signaling, which in turn plays roles in segmentation and neuroblast specification. In porc zygotic mutants, the BP102⁺ axon scaffold is only slightly affected; however, the maternally contributed porc mRNA may mask a requirement for porc in Wnt5 signaling in the zygotic porc mutant embryo. The demonstration of a requirement for porc in establishing the Wnt5 midline overexpression phenotype suggests that this assay will be a useful tool in uncovering novel members of the Wnt5 signaling pathway and its targets, which likely include cell surface adhesion proteins facilitating selective fasciculation and modulators of their activities (Fradkin, 2004).

GENE STRUCTURE

cDNA clone length - 3864 bp

Bases in 5' UTR - 551

Exons - 1

Bases in 3' UTR - 298

PROTEIN STRUCTURE

Amino Acids - 1004

Structural Domains

Wnt5 (also called Dwnt3), a member of the Wnt gene family in Drosophila. Wnt5 is a single-exon gene encoding an unusually large Wnt protein of 1,004 amino acids with a unique amino-terminal domain that seems to be proteolytically cleaved, followed by the Wnt domain common to all members of the family (Yoshikawa, 2003).

date revised: 7 Sept 2007

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.